Process for preparation of R-(-)-3-halogeno-1,2,-propanediol by treatment with microorganism

ABSTRACT

A process for preparation of R-(-)-3-halogeno-1,2-propanediol which comprises cultivating in a medium containing racemate 3-halogeno-1,2-propanediol a bacterium, which, when cultivated in a medium containing racemate 3-halogeno-1,2-propanediol as a sole carbon source, can grow and proliferate, has an ability to assimilate S-(+)-3-halogeno-1,2-propanediol preferentially compared to R-(-)-3-halogeno-1,2-propanediol and belongs to the genus Pseudomonas, or its culture cells; and recovering R-(-)-3-halogeno-1,2-propanediol from the resulting culture broth.

This application is a continuation of application Ser. No. 07/630,046,filed Dec. 19, 1990, now abandoned.

DETAILED DESCRIPTION OF THE INVENTION

1. Industrially Applicable Field

This invention relates to a process for separatively obtaining opticallyactive R-(-)-3-halogeno-1,2-propanediol from racemate3-halogeno-1,2-propanediol using a microorganism.

This invention further relates to a pure culture of a bacteriumbelonging to the genus Pseudomonas having the ability to assimilateS-(+)-3-halogeno-1,2-propanediol preferentially compared toR-(-)-3-halogeno-1,2-propanediol.

2. Prior Art

R-(-)-3-halogeno-1,2-propanediol is a useful substance as a raw materialfor synthesis of optically active pharmaceuticals and physiologicallyactive substances. R-(-)-3-halogeno-1,2-propanediol can, for example, beutilized for synthesis of optically active beta-adrenergic blockers,L-carnitine and the like. Further, R-(-)-3-halogeno-1,2-propanediol canbe converted to R-(+)-glycidol, a useful intermediate for synthesis ofoptically active pharmaceuticals and pesticides as well asphysiologically active substances and Ferroelectric Liquid Crystals.

As processes for preparation of this usefulR-(-)-3-halogeno-1,2-propanediol the following processes have hithertobeen known.

The process of Hayan F. Jones to obtain it frommethyl-5-chloro-5-deoxy-alpha-L-arabinofuranoside (Chemistry andIndustry, 15, P533, 1978) and the process of H. Jackson, et al. toobtain it from 1,2,5,6-diacetonyl-D-mannitol (Chem.-Biol. Interaction,13, P193, 1976) are chemical synthetic processes, and, in both cases,the raw materials are hard to obtain, high synthetic technique isnecessitated and the steps are complicated, and thus these processes areindustrially unsuitable.

On the other hand, Takahashi et al. proposed a process to obtainoptically active 3-chloro-1,2-propanediol using a microorganism(European Laid-Open Patent Publication Nos. 0224246 and 0286059).

In this process of using a microorganism, extremely many kinds ofmicroorganisms are proposed and several microorganisms belonging to thegenus Pseudomonas are included among them. These processes mainlyutilize the reaction that S-(+)-3-halogeno-1,2-propanediol isoxidatively decomposed and metabolized by action of a microorganism.However, these processes have the following problems.

Although Pseudomonas fragi IFO 3458, Pseudomonas crucicuiae IFO 12047and Pseudomonas chlororaphis IFO 3904 disclosed in the above EuropeanLaid-Open Patent Publication No. 0224246 can metabolizeS-(+)-3-halogeno-1,2-propanediol, as is the case with othermicroorganisms, they cannot grow and proliferate in a completelysynthetic medium containing racemate 3-halogeno-1,2-propanediol as asole carbon source and an inorganic nitrogen such as ammonium sulfate orammonium nitrate as a nitrogen source.

Therefore, when reaction is carried out utilizing any of the abovemicroorganisms, the process must be adopted which comprisesproliferating the cells in advance in a nutrient medium to obtain lotsof cells and making the cells after washing act on racemate3-halogeno-1,2-propanediol, as disclosed in the examples of the abovepatent. If the above process is not adopted, it becomes necessary to addracemate 3-halogeno-1,2-propanediol in a medium which contains othernutrients and wherein the microorganism can grow, and this latterprocess is not a preferred process in view of reaction efficiency orpurification of the product.

Problem to be Solved by the Invention

Thus, the object of the invention is to provide a process capable ofpreparing highly pure R-(-)-3-halogeno-1,2-propanediol economically,inexpensively and technically simply, compared to the aforesaid usualprocesses.

Means for Solving the Problem

As a result of vigorous studies to find a microorganism which canassimilate S-(+)-3-halogeno-1,2-propanediol preferentially compared toR-(-)-3-halogeno-1,2-propanediol and can grow and proliferate whencultivated in a medium containing racemate 3-halogeno-1,2-propanediol asa sole carbon source, the present inventors succeeded in isolation fromthe soil a microorganism capable of attaining the above object of theinvention and accomplished the invention.

Thus, the invention provides a process for preparation ofR-(-)-3-halogeno-1,2-propanediol which comprises cultivating in a mediumcontaining racemate 3-halogeno-1,2-propanediol a bacterium, which, whencultivated in a medium containing racemate 3-halogeno-1,2-propanediol asa sole carbon source, can grow and proliferate, has an ability toassimilate S-(+)-3-halogeno-1,2-propanediol preferentially compared toR-(-)-3-halogeno-1,2-propanediol and belongs to the genus Pseudomonas,or its culture cells; and recovering R-(-)-3-halogeno-1,2-propanediolfrom the resulting culture broth.

According to the process of the invention, even when mass production orR-(-)-3-halogeno-1,2-propanediol is undertaken, there is no need tocultivate a large number of cells separately and collect the resultingcells, and it is sufficient to cultivate the bacterium in an amountenough as a seed strain and inoculate it into the medium.

In assimilation of S-(+)-3-halogeno-1,2-propanediol by any of thebacteria which can be used in the invention and belong to the genusPseudomonas, dehydrohalogenation reaction ofS-(+)-3-halogeno-1,2-propanediol is carried out and as a resulthydrohalogenic acid is formed.

Suitable as 3-halogeno-1,2-propanediols used in the invention are3-chloro-1,2-propanediol and 3-bromo-1,2-propanediol.

The morphological and physiological properties of the threerepresentative bacteria, which were isolated and recovered from the soilby the inventors and can be used in the invention, are set forth belowin Table 1.

                                      TABLE 1                                     __________________________________________________________________________                       Pseudomonas sp.                                                                          Pseudomonas sp.                                                                        Pseudomonas sp.                                           DS-K-2D1   DS-K-9D1 DS-K-14A4                              __________________________________________________________________________    A. Morphology                                                                 1. Shape of cells  rods       same as left                                                                           same as left                           2. Size of cells   0.4-0.6 × 1.2-1.5 μm                                                            same as left                                                                           same as left                           3. Pleomorphisms cells                                                                           none       same as left                                                                           same as left                           4. Mobility        +, polar flagella                                                                        same as left                                                                           same as left                           5. Spores          none       same as left                                                                           same as left                           6. Gram stain      negative   same as left                                                                           same as left                           7. Acid fastness   none       same as left                                                                           same as left                           B. Growth condition in various media                                          1. Nutrient agar (3 days at 30° C.)                                    1) Speed of colony growth                                                                        ordinary   same as left                                                                           same as left                           2) Shape of colonies                                                                             circular   same as left                                                                           same as left                           3) Shape of colony surface                                                                       smooth     same as left                                                                           same as left                           4) Raised condition of colonies                                                                  convex     same as left                                                                           same as left                           5) Periphery of colonies                                                                         entire     same as left                                                                           same as left                           6) Contents of colonies                                                                          homogeneous                                                                              same as left                                                                           same as left                           7) Color of colonies                                                                             milky white                                                                              same as left                                                                           same as left                           8) Gloss of colonies                                                                             dull       same as left                                                                           same as left                           9) Transparency of colonies                                                                      translucent                                                                              same as left                                                                           same as left                           10)                                                                              Formation of soluble pigments                                                                 none       same as left                                                                           same as left                           2. Slant culture of nutrient agar                                                (for 3 days at 30° C.)                                              1) Growth degree   good       same as left                                                                           same as left                           2) Growth condition                                                                              filiform   same as left                                                                           same as left                           3) Shape of colony surface                                                                       smooth     same as left                                                                           same as left                           4) Shape of colonies in section                                                                  flat       same as left                                                                           same as left                           5) Gloss of colonies                                                                             dull       same as left                                                                           same as left                           6) Color tone of colonies                                                                        milky white                                                                              same as left                                                                           same as left                           7) Transparency of colonies                                                                      translucent                                                                              same as left                                                                           same as left                           3. Nutrient liquid standing culture                                              (for 3 days at 30° C.)                                              1) Growth condition                                                                              somewhat turbid                                                                          same as left                                                                           same as left                           2) Gas production  none       same as left                                                                           same as left                           3) Coloring of the medium                                                                        none       same as left                                                                           same as left                           4. Gelatin liquefaction test                                                                     -          -        -                                         (+: liquefacts gelatin,                                                       -: liquefacts no gelatin)                                                  5. Litmus milk (+: reduces litmus                                                                +          -        +                                         to white, non-coagulated,                                                     -: no change, non-coaculated)                                              6. MGPB agar (for 6 days                                                         at 30° C.) (MGPB agar:                                                 3-halogeno-1,2-propanediol 1.0%,                                              peptone 0.1%, yeast extract 0.1%,                                             Bromothymol Blue 0.01%,                                                       agar 2.0%, pH 7.0)                                                         1) Spped of colony growth                                                                        slow       same as left                                                                           same as left                           2) Shape of colonies                                                                             circular   same as left                                                                           same as left                           3) Shape of colony surface                                                                       smooth     wrinkled smooth                                                               (radial)                                        4) Raised condition of colonies                                                                  uumbonate  convex   convex                                 5) Periphery of colonies                                                                         entire     same as left                                                                           same as left                           6) Contents of colonies                                                                          homogeneous                                                                              same as left                                                                           same as left                           7) Color of colonies                                                                             orange at the                                                                            same as left                                                                           same as left                                              center                                                                        white at the                                                                  periphery                                                  8) Gloss of colonies                                                                             dull       same as left                                                                           same as left                           9) Transparency of colonies                                                                      opaque     same as left                                                                           same as left                           10)                                                                              Formation of soluble pigments                                                                 none       same as left                                                                           same as left                           C. Physiological test                                                         1. Lysine decarboxylation test                                                                   +           +       +                                      2. VP test         -          -        -                                      3. MR test         -          -        -                                      4. Reduction of nitrate                                                                          -          -        -                                      5. Production of indole                                                                          -          -        -                                      6. PPA reaction    -          -        -                                      7. Formation of hydrogen sulfide                                                                 -          -        -                                      8. Utilization of citric acid                                                                    +          +        +                                      9. Starch decomposition test                                                                     -          -        -                                      10.                                                                              Denitrification reaction                                                                      -          -        -                                         Utilization of inorganic salt                                                                 +          +        +                                         Formation of dye                                                              1) King A medium                                                                              -          -        -                                         2) King B medium                                                                              -          -        -                                         3) Pseudomonas P medium                                                                       -          -        -                                         4) Peseudomonas F medium                                                                      -          -        -                                         Catalase        +          +        +                                         Oxidase         +          +        +                                         Arginine dehydrogenase                                                                        -          -        -                                         Urease test     -          -        -                                         OF-test (Hugh Leifson method.                                                 No gas formation was observed.)                                               1) D-glucose    O          O        O                                         2) Glycerol     O          O        O                                         3) D-galactose  O          O        O                                         4) D-fructose   O          O        O                                         5) D-trehalose  O          O        O                                         Accumulation of PHB                                                                           +          +        +                                         Utilization of carbon sources                                                 1) D-mannitol   +          +        +                                         2) D-fructose   +          +        +                                         3) D-glucose    +          +        +                                         4) D-gluconic acid                                                                            +          +        +                                         5) D-galactose  +          +        +                                         6) Glycerol     +          +        +                                         7) p-Hydroxybenzoic acid                                                                      +          +        +                                      __________________________________________________________________________

By classification based on the results of Table 1 according to Bergey'sManual of Systematic Bacteriology 9th edition, it was revealed that allthe above strains belong to the genus Pseudomonas because they areGram-negative aerobic rods, have polar flagella, and areoxidase-positive and catalase-positive. Although there can be mentionedas their closely related strains Pseudomonas acidoborans, Pseudomonastestosteroni, Pseudomonas delafieldii, Pseudomonas facilis, Pseudomonassaccharophila, Pseudomonas flava, Pseudomonas pseudoflava andPseudomonas palleronii, the above strains differs from Pseudomonasacidoborans and Pseudomonas testosteroni in that all the former strainscan utilize D-glucose as a carbon source whereas the latter strainscannot utilize D-glucose as a carbon source, from Pseudomonasdelafieldii, Pseudomonas facilis, Pseudomonas saccharophila andPseudomonas flava in that all the former strains can utilizep-hydroxybenzoate as a carbon source whereas the latter strains cannotutilize p-hydroxybenzoate as a carbon source, and from Pseudomonaspseudoflava and Pseudomonas palleronii in that all the former strainsform no carotenoid pigment whereas the latter strains form carotenoidpigments. Thus, all the above strains do not accord with the knownstrains in characteristics, and were believed to be novel strains andnamed Pseudomonas sp. DS-K-2D1, Pseudomonas sp. DS-K-9D1 and Pseudomonassp. DS-K-14A4.

Further, although these strains are mutually analogous strains eachbelonging to the genus Pseudomonas, Pseudomonas sp. DS-K-2D1 andPseudomonas sp. DS-K-14A4 differ from Pseudomonas sp. DS-K-9D1 in thatthe former strains reduce litmus milk to white whereas the later straindoes not reduce litmus milk to white, and Pseudomonas sp. DS-K-2D1differs from Pseudomonas sp. DS-K-14A4 in that, in the growth conditionin the MGPB agar, the raised condition of colonies is convex at thecenter in the former whereas the raised condition of colonies is convexin the latter. Thus, the above strains were found as three mutuallydifferent strains.

The above three strains were deposited with Fermentation ResearchInstitute, Agency of Industrial Science and Technology, Japan on Nov.15, 1989, and, thereafter, their control was transferred in theFermentation Research Institute on Sep. 12, 1990 in accordance withBudapest Treaty on the international recognition of the deposit ofmicroorganism for the purpose of patent procedure. Deposit numbers ofthe respective strains are set forth below.

    ______________________________________                                               Pseudo-   Pseudo-     Pseudo-                                                 monas sp. monas sp.   monas sp.                                               DS-K-2D1  DS-K-9D1    DS-K-14A4                                        ______________________________________                                        FRI      FERM P-11109                                                                              FERM P-11108                                                                              FERM P-11110                                 (Nov. 15,                                                                     1989)                                                                         FRI      FERM BP-3096                                                                              FERM BP-3095                                                                              FERM BP-3097                                 under                                                                         Budapest                                                                      Treaty                                                                        (Sep. 12,                                                                     1990)                                                                         ______________________________________                                    

Strains which can be used in the invention and belong to the genusPseudomonas are not limited to the above three strains. Namely, otherstrains can similarly be used so long as they have an ability toassimilate S-(+)-3-halogeno-1,2-propanediol preferentially compared toR-(-)-3-halogeno-1,2-propanediol and can grow and proliferate in amedium containing racemate 3-halogeno-1,2-propanediol as a sole carbonsource.

Specifically, the process of the invention can be carried out either bycultivating any of the above bacteria in a synthetic medium containingracemate 3-halogeno-1,2-propanediol as a substantially sole carbonsource, inorganic nitrogens (various ammonium salts or nitrates) asnitrogen sources and further inorganic salts, and recovering theremaining R-(-)-3-halogeno-1,2-propanediol from the culture broth, or bycultivating any one of the above bacteria in a medium containing carbonsources, nitrogen sources, organic nutrient sources and inorganicsources and usually frequently used, for example, a bouillon medium or apeptone medium, inoculating the thus obtained culture broth or culturecells into a medium containing racemate 3-halogeno-1,2-propanediol as asole carbon source, further carrying out cultivation or making the cellsact, and then recovering the remaining R-(-)-3-halogeno-1,2-propanediolfrom the culture broth.

As carbon sources, there can be used, besides racemate3-halogeno-1,2-propanediol, alcohols such as glycerol, saccharides suchas D-glucose, D-fructose and D-galactose, and organic acids such ascitric acid, maleic acid, malic acid and fumaric acid and their salts.

It is advantageous in the process of the present invention to useracemate 3-halogeno-1,2-propanediol as the substantial carbon source.

Further, as nitrogen sources there can be used those usually used incultivation of cells. Examples thereof include inorganic nitrogens suchas ammonium sulfate, ammonium nitrate and ammonium phosphate, andorganic nitrogens such as urea, peptone, casein, yeast extract, meatextract and corn steep liquor. There can further be used generally knowninorganic salts such as phosphates, magnesium salts, potassium salts,manganese salts, zinc salts and copper salts.

Cultivation of the invention can be carried out by conventionalconditions and means. Namely, the cultivation is carried out at acultivation temperature of about 20° to 40° C., preferably 25° to 37°C., at a pH of about 5 to 10, preferably 5.5 to 9.5 and under an aerobiccondition, using a means of shaking cultivation or aeration stirringcultivation on the like. The substrate concentration in the reactionmixture is preferably on the order of 0.1 to 10% (v/v), and although thereaction time varies depending on the substrate concentration and otherreaction conditions, it is preferably 48 to 80 hours. It is preferredthat the reaction is discontinued at the time when the remainingsubstrate analyzed by gas chromatography or the like was reduced up to50%, namely at the time when S-(+)-3-halogeno-1,2-propanediol wassubstantially assimilated. Recovery and purification of the remainingR-(-)-3-halogeno-1,2-propanediol can be carried out as follows. Namely,the purification and recovery can be carried out by separating theculture broth after completion of the cultivation into the microbialcells and the supernatant by centrifugation, a flocculant agent or thelike, adsorbing on active carbon R-(-)-3-halogeno-1,2-propanediolremaining in the supernatant, and then either eluting it with acetoneand then subjecting the eluate to vacuum distillation, or extracting itwith a solvent such as ethyl acetate and then subjecting the extract tovacuum distillation.

The S-(+)-3-halogeno-1,2-propanediol-assimilating strains belonging tothe genus Pseudomonas in the invention are novel, and their purecultures are also provided by the invention. As these strains, there canparticularly preferably be used those named Pseudomonas sp. DS-K-9D1,Pseudomonas sp. DS-K-2D1 and Pseudomonas sp. DS-K-14A4.

EXAMPLE

The invention is specifically described below by examples. % in theexamples indicates % by weight unless otherwise noted.

EXAMPLE 1

100 ml of a medium having the composition of

    ______________________________________                                        ammonium sulfate     0.5%                                                     disodium hydrogenphosphate                                                                         0.1%                                                     dipotassium hydrogenphosphate                                                                      0.1%                                                     sodium dihydrogenphosphate                                                                         0.2%                                                     magnesium sulfate     0.05%                                                   iron sulfate, copper sulfate                                                                       trace                                                    and manganese sulfate                                                         calcium carbonate     0.45%                                                   pH 6.8                                                                        ______________________________________                                    

poured in a 500-ml Sakaguchi flask was sterilized at 121° C. for 15minutes, and then racemate 3-chloro-1,2-propanediol was added thereto to1.0% (v/v) to prepare a medium containing racemate3-chloro-1,2-propanediol as a sole carbon source. One platinum loopfulof a slant agar culture of Pseudomonas sp. DS-K-2Dl, one of themicroorganisms shown in Table 1, was inoculated into the above mediumand cultivated with shaking for 4 days under the conditions of 30° C.and 130 rpm.

After completion of the cultivation, the culture broth was taken out andsubjected to centrifugation to remove the cells and obtain thesupernatant. This supernatant was concentrated up to about 20 ml and theconcentrate was extracted with ethyl acetate. The extract was dehydratedwith anhydrous magnesium sulfate and kept under reduced pressure toobtain 0.4 g of 3-chloro-1,2-propanediol as an oily substance.

Identification of this substance was carried out by gas chromatography.When the substance was compared with commercially available racemate3-chloro-1,2-propanediol (product of Tokyo Kasei Co.) using a columncarrier PEG-20MP, 60-80 mesh, the retention times of both substanceswere utterly identical.

The specific rotation of the present substance and the specific rotationof (R)-3-chloro-1,2-propanediol (literature value) are as follows.

    ______________________________________                                        The present substance                                                                             [α].sub.D.sup.22 = -7.75°                                        (C = 1, H.sub.2 O)                                        Literature value    [α].sub.D.sup.22 = -6.9°                                         (C = 2, H.sub.2 O)                                        ______________________________________                                    

Further, after the present substance was tosylated according to aconventional method, HPLC analysis was carried out under the conditionsof room temperature, a flow rate of 1.0 ml/min and a wavelength of 235nm using an optical isomer-separating column [CHIRALCEL OC column (25cm×0.46 cm I.D.)] (produced by DAICEL CHEMICAL INDUSTRIES, LTD.) andhexane-isopropanol (95:5) as a solvent. Retention times by this analyticmethod are 79 minutes for the S form compound and 89.8 minutes for the Rform compound, and the present substance exhibited a retention timecorresponding to that of the R form compound and its enantiomer excesswas 98% e.e. or more.

EXAMPLES 2 AND 3

The same operations as in Example 1 were carried out except that thestrain was replaced by Pseudomonas sp. DS-K-9D1 or Pseudomonas sp.DS-K-14A4. When analyzed according to the various analytical methods asshown in Example 1, each of the obtained substances was revealed to beR-(-)-3-chloro-1,2-propanediol having an enantiomer excess of 98% e.e.or more. The results of Examples 2 and 3 are shown together below.

    ______________________________________                                        Example               [α].sub.D.sup.22                                  No.       Strain      (C = 1, H.sub.2 O)                                                                       Yield (g)                                    ______________________________________                                        2         DS-K-9D1    -7.70°                                                                            0.38                                         3         DS-K-14A4   -7.71°                                                                            0.45                                         ______________________________________                                    

EXAMPLE 4

2.5 l of the medium having the composition of

    ______________________________________                                        ammonium sulfate     0.5%                                                     disodium hydrogenphosphate                                                                         0.02%                                                    dipotassium hydrogenphosphate                                                                      0.02%                                                    sodium dihydrogenphosphate                                                                         0.04%                                                    magnesium sulfate    0.05%                                                    iron sulfate, copper sulfate                                                                       trace                                                    and manganese sulfate                                                         pH 6.8                                                                        ______________________________________                                    

poured in a 5-liter cultivation apparatus (jar fermentor) was sterilizedat 121° C. for 15 minutes, and then racemate 3-chloro-1,2-propanediolwas added thereto to 1.0% (v/v) to prepare a medium containing racemate3-chloro-1,2-propanediol as a sole carbon source. Then, Pseudomonas sp.DS-K-2D1, one of the microorganisms indicated in Table 1, was previouslycultivated with shaking at 30° C. for 24 hours in a nutrient mediumcomprising 1.0% of peptone, 1.0% of yeast extract and 1.0% of glycerol,pH 7.2, and the culture broth was aseptically inoculated into the abovemedium containing racemate 3-chloro-1,2-propanediol as a sole carbonsource so that its amount became 2% (v/v). Aerobic stirring cultivationwas carried out for 4 days under the following conditions:

    ______________________________________                                        Temperature           30° C.                                           Aeration amount       0.5 1/min                                               agitation             500 rpm                                                 ______________________________________                                    

Measurement and control of pH were carried out by a linked pH meter andthe pH was controlled at 6.8 with 3N-NaOH.

After completion of the cultivation, the culture broth was taken out andsubjected to centrifugation to remove the cells and obtain thesupernatant. The supernatant was treated in the same manner as inExample 1 to obtain 14.9 g of 3-chloro-1,2-propanediol. The obtainedsubstance was subjected to the various analyses as shown in Example 1,and as a result it was revealed that the substance wasR-(-)-3-chloro-1,2-propanediol having an enantiomer excess of 98% e.e.or more. The specific rotation [α]_(D) ²² of this substance was -7.75°(C=1, H₂ O).

EXAMPLES 5 and 6

The same operations as in Example 4 were carried out except that thestrain was replaced by Pseudomonas sp. DS-K-9D1 or Pseudomonas sp.DS-K-14A4 shown in Table 1. By the various analyses shown in Example 1,each of the obtained substances was revealed to beR-(-)-3-chloro-1,2-propanediol having an enantiomer excess of 98% e.e.or more. The results of Examples 5 and 6 are shown below in a lump.

    ______________________________________                                        Example               [α].sub.D.sup.22                                  No.       Strain      (C = 1, H.sub.2 O)                                                                       Yield (g)                                    ______________________________________                                        5         DS-K-9D1    -7.74°                                                                            15.0                                         6         DS-K-14A4   -7.70°                                                                            13.5                                         ______________________________________                                    

EXAMPLE 7

100 ml of a nutrient medium having the composition of 1.0% of peptone,1.0% of yeast extract and 1.0% of glycerol, pH 7.2 in a 500-ml Sakaguchiflask was sterilized at 121° C. for 15 minutes, and then one platinumloopful of a slant agar culture of Pseudomonas sp. DS-K-2D1 shown inTable 1 was inoculated thereinto. Cultivation was carried out at 30° C.for 24 hours with shaking, the cells and the supernatant were separatedby centrifugation and the supernatant was discarded. The resulting cellswere washed a few times with a 50 mM phosphate buffer, pH 7.0. The cellswere then suspended in 100 ml of the medium containing racemate3-chloro-1,2-propanediol as a sole carbon source shown in Example 1, andreaction was carried out at 30° C. and 130 rpm for 3 days. Aftercompletion of the reaction, the cells were removed by centrifugation toobtain the supernatant. The supernatant was treated in the same manneras in Example 1 to obtain 0.4 g of 3-chloro-1,2-propanediol. Thesubstance was subjected to the various analyses as shown in Example 1,and as a result it was revealed that the substance wasR-(-)-3-chloro-1,2-propanediol having an enantiomer excess of 98% e.e.or more. The specific rotation [α]_(D) ²² of the substance was -7.75°(C=1, H₂ O).

EXAMPLES 8 AND 9

The same operations as in Example 7 were carried out except that thestrain was replaced by Pseudomonas sp. DS-K-9D1 or Pseudomonas sp.DS-K-14A4 shown in Table 1. By the various analyses as shown in Example1, each of the obtained substances was revealed to beR-(-)-3-chloro-1,2-propanediol having an enantiomer excess of 98% e.e.or more. The results of Examples 8 and 9 are shown below in a lump.

    ______________________________________                                        Example               [α].sub.D.sup.22                                  No.       Strain      (C = 1, H.sub.2 O)                                                                       Yield (g)                                    ______________________________________                                        8         DS-K-9D1    -7.74°                                                                            0.51                                         9         DS-K-14A4   -7.70°                                                                            0.45                                         ______________________________________                                    

EXAMPLES 10 to 12

The same experiments as in Examples 1 to 3 were carried out except thatracemate 3-chloro-1,2-propanediol was replaced by racemate3-bromo-1,2-propanediol. Experimental methods and other operations werecarried out according to Example 1. The results of Examples 10 to 12 areshown below in a lump.

    ______________________________________                                        Example               [α].sub.D.sup.22                                  No.       Strain      (C = 1, H.sub.2 O)                                                                       Yield (g)                                    ______________________________________                                        10        DS-K-2D1    -3.80°                                                                            0.61                                         11        DS-K-9D1    -3.75°                                                                            0.65                                         12        DS-K-14A4   -3.77°                                                                            0.55                                         ______________________________________                                    

In this connection, the literature value of the specific rotation of(R)-3-bromo-1,2-propanediol is as follows:

    [α].sub.D.sup.25 =-3.94° (C=5.07, CHCl.sub.3).

Further, each of R-(-)-3-bromo-1,2-propanediols obtained by Examples 10to 12 was tosylated by a conventional manner, and the tosylated compoundwas subjected to HPLC analysis under the conditions of room temperature,a flow rate of 1.0 ml/min and a wavelength of 235 nm using an opticalisomer-separating column [CHIRALCEL, OC column (25 cm×0.46 cm I.D.)](produced by DAICEL CHEMICAL INDUSTRIES, LTD.) and hexane-isopropanol(95:5) as a solvent. Retention times in this analysis are 98.9 minutesfor the S-form compound and 115.4 minutes for the R-form compounds, andeach of the above substances exhibited a retention time corresponding tothe R-form compound and had an enantiomer excess of 96% e.e. or more.

EXAMPLES 13 to 15

Experiments were carried out in accordance with Examples 4 to 6 exceptthat racemate 3-chloro-1,2-propanediol was replaced by racemate3-bromo-1,2-propanediol. Experimental methods and other operations werecarried out according to Example 4. By the various analyses as shown inExample 10, each of the obtained substances was revealed to beR-(-)-3-bromo-1,2-propanediol having an enantiomer excess of 96% e.e. ormore. The results of Examples 13 to 15 are shown below in a lump.

    ______________________________________                                        Example               [α].sub.D.sup.22                                  No.       Strain      (C = 1, H.sub.2 O)                                                                       Yield (g)                                    ______________________________________                                        13        DS-K-2D1    3.77°                                                                             17.1                                         14        DS-K-9D1    -3.74°                                                                            15.5                                         15        DS-K-14A4   -3.78°                                                                            16.8                                         ______________________________________                                    

EXAMPLES 16 to 18

Experiments were carried out in accordance with Examples 7 to 9 exceptthat racemate 3-chloro-1,2-propanediol was replaced by racemate3-bromo-1,2-propanediol. Experimental methods and other operations werecarried out according to Example 7. By the various analyses as shown inExample 10, each of the obtained substances was revealed to beR-(-)-3-bromo-1,2-propanediol having an enantiomer excess of 96% e.e. ormore. The results of Examples 16 to 18 are shown below in a lump.

    ______________________________________                                        Example               [α].sub.D.sup.22                                  No.       Strain      (C = 1, H.sub.2 O)                                                                       Yield (g)                                    ______________________________________                                        16        DS-K-2D1    -3.77°                                                                            0.62                                         17        DS-K-9D1    -3.78°                                                                            0.59                                         18        DS-K-14A4   -3.74°                                                                            0.66                                         ______________________________________                                    

Effects of the Invention

In accordance with the invention, it is possible to prepareR-(-)-3-halogeno-1,2-propanediol by a method wherein inexpensive rawmaterials can be utilized and which is industrially convenient, namelyby assimilating S-(+)-3-halogeno-1,2-propanediol preferentially fromracemate 3-halogeno-1,2-propanediol utilizing a bacterium of the genusPseudomonas.

We claim:
 1. A process for preparation of R-(-)-3-chloro-1,2-propanediolor R-(-)-3-bromo-1,2-propanediol which comprises cultivating andproliferating, in a medium containing racemate 3-chloro-1,2-propanediolor racemate 1-bromo-1,2-propanediol as an essential carbon source, abacterium belonging to the genus Pseudomonas which is selected from thegroup consisting of Pseudomonas sp. DS-K-9D1, Pseudomonas sp. DS-K-2D1and Pseudomonas sp. DS-K-14A4 which are deposited with FermentationResearch Institute, Agency of Industrial Science and Technology, Japanas Deposit Nos. FERM BP-3095, FERM BP-3096 and FERM BP-3097,respectively, or its culture cell, and recoveringR-(-)-3-chloro-1,2-propanediol or R-(-)-3-bromo-1,2-propanediol from theresulting culture broth.
 2. The process of claim 1 in which each of saidbacteria has the ability to grow and proliferate when cultivated in amedium containing racemate 3-chloro-1,2-propanediol or racemate3-bromo-1,2-propanediol as the sole carbon source.
 3. The process ofclaim 1 for preparing R-(-)-3-chloro-1,2-propanediol wherein the mediumcontains racemate 3-chloro-1,2-propanediol.
 4. The process of claim 1for preparing R-(-)-3-chloro-1,2-propanediol wherein the medium containsracemate 3-bromo-1,2-propanediol.
 5. The process of claim 1 wherein thecultivation is carried out under an aerobic condition.
 6. The process ofclaim 1 wherein the cultivation is carried out at a temperature of about20° C. to about 40° C.
 7. The process of claim 1 wherein the cultivationis carried out at a pH of about 5 to about
 10. 8. The process of claim 1wherein the bacterium is Pseudomonas sp. DS-K-9D1.
 9. The process ofclaim 1 wherein the bacterium is Pseudomonas sp. DS-K-2D1.
 10. Theprocess of claim 1 wherein the bacterium is Pseudomonas sp. DS-K-14A4.11. The process of claim 1 wherein the cultivation is carried out underaerobic conditions at a temperature of from about 25° to 37° C. at a pHof from about 5.5 to 9.5 at a substrate concentration in the reactionmixture of from 0.1 to 10% (v/v) for from about 48 to 80 hours.
 12. Theprocess of claim 11 wherein the cultivation is carried out in a mediumcontaining racemate 3-chloro-1,2-propanediol as the sole carbon source.13. The process of claim 1 in which the cultivation is carried out in amedium containing racemate 3-bromo-1,2-propanediol as the sole carbonsource.